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The primary aim of this randomized trial was to determine response rate and progression-free survival following chemotherapy in patients with platinum-resistant recurrent ovarian cancer, who had been treated according to an ATP-based tumour chemosensitivity assay in comparison with physician's choice. A total of 180 patients were randomized to assay-directed therapy (n=94) or physician's-choice chemotherapy (n=86). Median follow-up at analysis was 18 months. Response was assessable in 147 patients: 31.5% achieved a partial or complete response in the physician's-choice group compared with 40.5% in the assay-directed group (26 versus 31% by intention-to-treat analysis respectively). Intention-to-treat analysis showed a median progression-free survival of 93 days in the physician's-choice group and 104 days in the assay-directed group (hazard ratio 0.8, 95% confidence interval 0.59-1.10, not significant). No difference was seen in overall survival between the groups, although 12/39 (41%) of patients who crossed over from the physician's-choice arm obtained a response. Increased use of combination therapy was seen in the physician's-choice arm during the study as a result of the observed effects of assay-directed therapy in patients. Patients entering the physician's-choice arm of the study during the first year did significantly worse than those who entered in the subsequent years (hazard ratio 0.44, 95% confidence interval 0.2-0.9, P<0.03). This small randomized clinical trial has documented a trend towards improved response and progression-free survival for assay-directed treatment. Chemosensitivity testing might provide useful information in some patients with ovarian cancer, although a larger trial is required to confirm this. The ATP-based tumour chemosensitivity assay remains an investigational method in this condition.

Due to its pivotal role in signal transduction, the universal tumor suppressor PTEN (also termed MMAC or TEP) is one of the putative candidates for involvement in tumorigenesis of several tissues. Although involvement of PTEN in tumorigenesis was shown in different tissues, no data are available concerning PTEN activity in response to antineoplastic agents. Therefore, we assayed the PTEN activity exposed to either blank medium or the commonly used anti-cancer drugs cisplatin, adriamycin or paclitaxel, respectively, in three different concentrations. PTEN activity was determined using the Malachite Green assay basing upon dephosphorylation of phosphatidylinositol-3,4,5-triphosphate (PIP3) by the PTEN enzyme and subsequent determination of inorganic phosphate released. Although the three different anti-cancer drugs assayed act with different cellular modes, the antineoplastics influenced PTEN activity in a similar manner: at low concentrations tested all three antineoplastics significantly increased PTEN activity. However, increasing drug concentrations exhibited a decline but not a total loss of PTEN activity. The data indicate that PTEN activity is increased following cytotoxic drug exposure and, thereby, exhibits its suppressive function. However, the decrease of PTEN activity in response to increasing drug concentrations suggests an aberration of total functional activity. As far as the regulative checkpoint PTEN is abolished, tumor cells might evade cell death pathways resulting in increased proliferation of cancer cells. This might be a general event in refractory tumor cells surviving chemotherapy.

This report describes preclinical and early clinical investigations of the mitoxantrone/paclitaxel combination (NT) for patients with platinum-refractory ovarian cancer. The preclinical activity of NT was studied ex vivo, evaluating native tumor specimens with the ATP tumor chemosensitivity assay. Of 24 tumors tested, 20 (83%) were sensitive to NT, whereas 7 (29%) responded to mitoxantrone and 8 (33%) responded to paclitaxel. In the majority of tumors assayed (19 of 24), potentiating or major independent effects between both agents were found. Subsequently, a clinical pilot trial of NT was initiated for patients with platinum-refractory ovarian cancer. Patients had failed one to four (median, two) prior chemotherapy regimens. In 11 cases, NT was administered every three weeks with 8 mg/m2 mito-xantrone and 180 mg/m2 paclitaxel (NT-I). Seven patients were treated biweekly with 6 mg/m2 mitoxantrone and weekly with 100 mg/m2 paclitaxel (NT-II). During 92 NT courses, myelosuppression with leucopenia, anemia, and thrombocytopenia was the limiting toxicity, occurring more frequently with NT-II. No patient required hospitalization due to any life-threatening complication. Five complete and nine partial remissions were observed with both NT-I and NT-II, accounting for an overall 78% response rate, with a median progression-free survival of 40 weeks. One patient showed early progression during therapy. Currently, three patients (NT-I, two; NT-II, one) have died due to progressive relapsed ovarian cancer, so that the median overall survival is not reached after a median follow-up of 40.5+ weeks. Both schedules were found to be equal in terms of response rate and overall survival. NT is highly active and practical for salvage treatment of ovarian cancer. NT-II may be preferred due to both clinical activity and patients' acceptance. However, NT-I seems to be a less myelotoxic alternative. Both schedules warrant further clinical investigation.

Utilizing a microplate ATP bioluminescence assay, two human breast carcinoma cell lines, MCF-7 and MDA-MB-231, were tested against doxorubicin (DOX), cisplatin (DDP), and paclitaxel (Tx) alone and in combination with ascorbic acid (Vit C). In both cell lines, Vit C exhibited cytotoxic activity at high concentrations (i.e. 10(2)-10(3) microM). Both cell lines also were resistant to DOX. MCF-7 was found to be DDP-resistant, MDA-MB-231 was moderately sensitive to DDP. Both cell lines were strongly sensitive to Tx. Vit C both at non-cytotoxic (1 microM) and moderately cytotoxic concentrations (10(2) microM) improved the cytotoxicity of DOX, DDP, and Tx significantly. Combination effects between Vit C and DDP or Tx were partly synergistic and partly additive or subadditive whereas a consistent synergism was found between Vit C and DOX. The mechanisms by which Vit C potentiates the cytostatics studied are yet unclear and should be evaluated further.

An ATP luminescence assay (TCA 100) was used to measure chemotherapeutic drug sensitivity and resistance of dissociated tumor cells cultured for 6 days in serum-free medium and 96-well polypropylene microplates. Studies were performed with surgical, needle biopsy, pleural, or ascitic fluid specimens using 10,000-20,000 cells/well. ATP measurements were used to determine tumor growth inhibition. Single agent and drug combinations were evaluated using the area under the curve and 50% inhibitory concentration (IC50) results for a series of test drug concentrations. The ATP luminometry method had high sensitivity, linearity, and precision for measuring the activity of single agents and drug combinations. Assay reproducibility was high with intraassay and interassay coefficients of variation of 10-15% for percentage of tumor growth inhibition, 5-10% for area under curve, and 15-20% for IC50 results. Good correlation (r = 0.93) between the area under the curve, and IC50 results was observed. Cytological studies with 124 specimens demonstrated selective growth of malignant cells in the serum-free culture system. Studies with malignant and benign specimens also showed selective growth of malignant cells in the serum-free medium used for assay. The assay had a success rate of 87% based on criteria for specimen histopathology, magnitude of cell growth, and dose-response drug activity. Cisplatin results for ovarian carcinoma are presented for 81 specimens from 70 untreated patients and 33 specimens from 30 refractory patients. A model for interpretation of these results based on the correlation of clinical response with the area under the curve and IC50 results indicates that the assay has > 90% accuracy for cisplatin resistance of ovarian carcinoma. Additional studies are in progress to evaluate the clinical efficacy of this assay.

Titanocenedichloride (MKT 4) is a novel anticancer drug with a broad spectrum of activity in mammalian tumors. We investigated the anticancer efficacy of MKT 4 versus cisplatin and its chemomodulation by buthionine sulfoximine (BSO) in four different human ovarian carcinoma (OvCA) cell lines derived from both primary (A2780. OTN 14) and recurrent tumors (SKOV-3 and OV-MZ-1b) using an in vitro microplate ATP bioluminescence assay (ATP-TCA). Sensitivity against cisplatin was higher in A2780 and OTN 14 compared with MKT 4, whereas the opposite was found in SKOV-3 and OV-MZ-1b cells. In A2780, SKOV-3 and OV-MZ-1b, the cytotoxicity of both agents could be effectively improved by BSO with supraadditive effects observed for MKT 4 in all three cell lines. In OTN 14, however, BSO treatment failed to increase the cytotoxicity of both cisplatin and MKT 4. These results suggest antineoplastic activity of MKT 4 in cisplatin-sensitive and mainly in cisplatin-resistant OvCA cells which can be significantly modulated by BSO-mediated glutathione depletion. Since antineoplastic activity of both cisplatin and MKT-4 observed in OTN 14 could not be reversed by BSO, other mechanisms of drug resistance different from the glutathione redox cycle are likely to be important for both metal compounds.

Background: Titanocenedichloride (MKT 4) is a new antineoplastic metal complex with proven activity in several experimental tumors., Material and Methods: In the present study, the cytotoxic activity of titanocenedichloride in fourteen primary and twelve recurrent ovarian carcinomas (OvCA) was evaluated by an in vitro adenosine triphosphate (ATP) bioluminescence assay., Results: In primary tumors, MKT 4 was found to be at least as effective as cisplatin (DDP) and doxorubicin (DOX). In samples derived from pretreated patients, titanocenedichloride was even more active. In both groups of tumors, a lack of cross resistance between the two metal compounds as well as between MKT 4 and DOX was apparent. The new agent was found to be active in eight of seventeen DDP-resistant (primaries: n = 4; recurrences n = 4) and also eight of seventeen DOX-resistant tumors (primaries: n = 4; recurrences n = 4)., Conclusions: These results indicate a remarkable in vitro activity of titanocenedichloride in native OvCA specimens, even in those exhibiting resistance against cisplatin or doxorubicin. The putative role of this novel drug for the future therapy of OvCA should be evaluated by additional in vitro and in vivo studies.